ABSTRACTHypoxia-inducible factors (HIFs) are master regulators of adaptive responses to low oxygen, and their α-subunits are rapidly degraded through the ubiquitination-dependent proteasomal pathway after hydroxylation. Aberrant accumulation or activation of HIFs is closely linked to many types of cancer. However, how hydroxylation of HIFα and its delivery to the ubiquitination machinery are regulated remains unclear. Here we show that Rho-related BTB domain-containing protein 3 (RHOBTB3) directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation. RHOBTB3 also directly interacts with the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin ligase complex, facilitating ubiquitination of HIFα. Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα. Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα. Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth. Our work thus reveals that RHOBTB3 serves as a scaffold to organize a multi-subunit complex that promotes the hydroxylation, ubiquitination and degradation of HIFα.

fig5: Dimerization of RHOBTB3 and LIMD1. (A) Ectopically expressed HA-tagged RHOBTB3 interacts with MYC-tagged RHOBTB3 or FLAG-tagged LIMD1. HEK293T cells were transfected with different combinations of HA-RHOBTB3, MYC-RHOBTB3 and FLAG-LIMD1. Cells were then lysed and the protein extracts were immunoprecipitated with antibody against HA. The IP product was analyzed by immunoblotting. (B) Ectopically expressed HA-tagged LIMD1 interacts with FLAG-tagged LIMD1 or MYC-tagged RHOBTB3. Lysates from transfected cells were subjected to IP with antibody against HA (for LIMD1), and analyzed by immunoblotting as in A. (C) RHOBTB3 interacts with endogenous LIMD1. Lysates of HEK293T cells were immunoprecipitated with antibody against RHOBTB3 or IgG (control), and analyzed by immunoblotting using antibodies indicated. (D) Knockdown of LIMD1 attenuates the interaction between RHOBTB3 and PHD2/VHL. HEK293T cells were infected with lentivirus expressing siRNA targeting GFP or LIMD1. At 16 h post-infection, cells were lysed and the endogenous RHOBTB3 was then immunoprecipitated, and analyzed by immunoblotting with antibodies indicated. (E, F) Ectopically expressed RHOBTB3 promotes the interaction between LIMD1 and PHD2 (E), and the interaction between LIMD1 and VHL (F). HEK293T cells were transfected with different combinations of HA-LIMD1, FLAG-RHOBTB3, MYC-PHD2 (E) and MYC-VHL (F). Protein extracts were immunoprecipitated and analyzed by immunoblotting. (G) Hypoxia attenuates the interaction between endogenous RHOBTB3/LIMD1 and PHD2/VHL. HEK293T cells were maintained in normoxia or exposed to hypoxia for 8 h in presence of 10 μM MG-132 to prevent the degradation of VHL under hypoxic condition as described previously80. Endogenous RHOBTB3 was then immunoprecipitated, and the IP product was analyzed by immunoblotting with antibodies indicated.

Mentions:
We were intrigued that RHOBTB3 and LIMD1, two proteins unrelated in sequence, could both enhance the interaction between PHD2 and VHL, and reduce HIFα in cells. We thus characterized the biochemistry of RHOBTB3 and LIMD1 in the context of HIF1α degradation. We found that ectopically expressed MYC-tagged RHOBTB3 interacted with HA-tagged RHOBTB3 (Figure 5A), suggesting that RHOBTB3 can form homodimers. Similarly, HA-tagged LIMD1 was co-precipitated with FLAG-tagged LIMD1 (Figure 5B), indicating that LIMD1 can homodimerize. More intriguingly, when co-expressed, RHOBTB3 could be co-precipitated with LIMD1 and vice versa, indicating that RHOBTB3 can form heterodimers with LIMD1 (Figure 5A, 5B and Supplementary information, Figure S6C). In addition, endogenous LIMD1 was readily co-immunoprecipitated with RHOBTB3 (Figure 5C). Moreover, the expression of MYC-RHOBTB3 disrupted the interaction between HA-LIMD1 and FLAG-LIMD1, leading to the formation of more LIMD1-RHOBTB3 heterodimers, and addition of FLAG-LIMD1 also reduced RHOBTB3 homodimerization (Figure 5A and 5B). These results suggest that RHOBTB3 and LIMD1 prefer to heterodimerize with each other, or form a higher order complex, than to homodimerize by themselves. Moreover, knockdown of RHOBTB3 or LIMD1 reduced the interaction between endogenous LIMD1/RHOBTB3 and PHD2/VHL (Figure 4E and 5D), whereas expression of RHOBTB3 strongly promoted the interaction between PHD2/VHL and LIMD1 (Figure 5E and 5F), suggesting that the RHOBTB3-LIMD1 heterodimers are more effective scaffolds for the formation of the HIFα degradation complexes that contain PHD2 and VHL. Furthermore, we observed that the interaction between PHD2 and VHL (Figure 3G), and the interaction between RHOBTB3-LIMD1 heterodimer and VHL/PHD2 were attenuated under hypoxic conditions (Figure 5G). The level of co-localization of RHOBTB3 and VHL was reduced under hypoxic condition (Supplementary information, Figure S3B). One obvious explanation is that RHOBTB3 and LIMD1 exert a tight control on the levels of HIF1α under normoxic condition, which is eased up in hypoxia to allow for appropriate HIFα accumulation. Notably, although LIMD1 and RHOBTB3 cooperatively regulate HIF1α levels, LIMD1, unlike RHOBTB3, does not promote the hydroxylation on the proline-564 residue of HIF1α as RHOBTB3 does (Supplementary information, Figure S6D and S6E).

fig5: Dimerization of RHOBTB3 and LIMD1. (A) Ectopically expressed HA-tagged RHOBTB3 interacts with MYC-tagged RHOBTB3 or FLAG-tagged LIMD1. HEK293T cells were transfected with different combinations of HA-RHOBTB3, MYC-RHOBTB3 and FLAG-LIMD1. Cells were then lysed and the protein extracts were immunoprecipitated with antibody against HA. The IP product was analyzed by immunoblotting. (B) Ectopically expressed HA-tagged LIMD1 interacts with FLAG-tagged LIMD1 or MYC-tagged RHOBTB3. Lysates from transfected cells were subjected to IP with antibody against HA (for LIMD1), and analyzed by immunoblotting as in A. (C) RHOBTB3 interacts with endogenous LIMD1. Lysates of HEK293T cells were immunoprecipitated with antibody against RHOBTB3 or IgG (control), and analyzed by immunoblotting using antibodies indicated. (D) Knockdown of LIMD1 attenuates the interaction between RHOBTB3 and PHD2/VHL. HEK293T cells were infected with lentivirus expressing siRNA targeting GFP or LIMD1. At 16 h post-infection, cells were lysed and the endogenous RHOBTB3 was then immunoprecipitated, and analyzed by immunoblotting with antibodies indicated. (E, F) Ectopically expressed RHOBTB3 promotes the interaction between LIMD1 and PHD2 (E), and the interaction between LIMD1 and VHL (F). HEK293T cells were transfected with different combinations of HA-LIMD1, FLAG-RHOBTB3, MYC-PHD2 (E) and MYC-VHL (F). Protein extracts were immunoprecipitated and analyzed by immunoblotting. (G) Hypoxia attenuates the interaction between endogenous RHOBTB3/LIMD1 and PHD2/VHL. HEK293T cells were maintained in normoxia or exposed to hypoxia for 8 h in presence of 10 μM MG-132 to prevent the degradation of VHL under hypoxic condition as described previously80. Endogenous RHOBTB3 was then immunoprecipitated, and the IP product was analyzed by immunoblotting with antibodies indicated.

Mentions:
We were intrigued that RHOBTB3 and LIMD1, two proteins unrelated in sequence, could both enhance the interaction between PHD2 and VHL, and reduce HIFα in cells. We thus characterized the biochemistry of RHOBTB3 and LIMD1 in the context of HIF1α degradation. We found that ectopically expressed MYC-tagged RHOBTB3 interacted with HA-tagged RHOBTB3 (Figure 5A), suggesting that RHOBTB3 can form homodimers. Similarly, HA-tagged LIMD1 was co-precipitated with FLAG-tagged LIMD1 (Figure 5B), indicating that LIMD1 can homodimerize. More intriguingly, when co-expressed, RHOBTB3 could be co-precipitated with LIMD1 and vice versa, indicating that RHOBTB3 can form heterodimers with LIMD1 (Figure 5A, 5B and Supplementary information, Figure S6C). In addition, endogenous LIMD1 was readily co-immunoprecipitated with RHOBTB3 (Figure 5C). Moreover, the expression of MYC-RHOBTB3 disrupted the interaction between HA-LIMD1 and FLAG-LIMD1, leading to the formation of more LIMD1-RHOBTB3 heterodimers, and addition of FLAG-LIMD1 also reduced RHOBTB3 homodimerization (Figure 5A and 5B). These results suggest that RHOBTB3 and LIMD1 prefer to heterodimerize with each other, or form a higher order complex, than to homodimerize by themselves. Moreover, knockdown of RHOBTB3 or LIMD1 reduced the interaction between endogenous LIMD1/RHOBTB3 and PHD2/VHL (Figure 4E and 5D), whereas expression of RHOBTB3 strongly promoted the interaction between PHD2/VHL and LIMD1 (Figure 5E and 5F), suggesting that the RHOBTB3-LIMD1 heterodimers are more effective scaffolds for the formation of the HIFα degradation complexes that contain PHD2 and VHL. Furthermore, we observed that the interaction between PHD2 and VHL (Figure 3G), and the interaction between RHOBTB3-LIMD1 heterodimer and VHL/PHD2 were attenuated under hypoxic conditions (Figure 5G). The level of co-localization of RHOBTB3 and VHL was reduced under hypoxic condition (Supplementary information, Figure S3B). One obvious explanation is that RHOBTB3 and LIMD1 exert a tight control on the levels of HIF1α under normoxic condition, which is eased up in hypoxia to allow for appropriate HIFα accumulation. Notably, although LIMD1 and RHOBTB3 cooperatively regulate HIF1α levels, LIMD1, unlike RHOBTB3, does not promote the hydroxylation on the proline-564 residue of HIF1α as RHOBTB3 does (Supplementary information, Figure S6D and S6E).

Bottom Line:
Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα.Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα.Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth.

ABSTRACTHypoxia-inducible factors (HIFs) are master regulators of adaptive responses to low oxygen, and their α-subunits are rapidly degraded through the ubiquitination-dependent proteasomal pathway after hydroxylation. Aberrant accumulation or activation of HIFs is closely linked to many types of cancer. However, how hydroxylation of HIFα and its delivery to the ubiquitination machinery are regulated remains unclear. Here we show that Rho-related BTB domain-containing protein 3 (RHOBTB3) directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation. RHOBTB3 also directly interacts with the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin ligase complex, facilitating ubiquitination of HIFα. Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα. Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα. Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth. Our work thus reveals that RHOBTB3 serves as a scaffold to organize a multi-subunit complex that promotes the hydroxylation, ubiquitination and degradation of HIFα.